1. Field of the Invention
The present invention relates to the general fields of biopolymer synthesis and reactions on surfaces of solid or soluble polymers, glass, gold, silica, metal oxide or other suitable materials (support). This invention particularly provides for chemical compounds to be used as capping agents for the termination of reactive groups on a support and the termination of reactive groups on the first layer of moieties from surface, chain, and/or intermediate sequences of a multiple step synthesis.
2. Description of the Background Art
Biopolymer synthesis on a support has been widely adopted for large-scale combinatorial synthesis, especially for oligonucleotides (Beaton, G. et al., Oligonucleotides and their Analogues, A Practical Approach, IRL Press, Oxford, UK, pp. 109-135 (1991)), peptides (Solid-phase peptide synthesis, Meth. Enzymol., Vol. 289, Academic Press: New York (1997)), and carbohydrates (Sears, P. et al., Toward Automated Synthesis of Oligosaccharides and Glycoproteins, Science 2350 (2001)). In these syntheses, it is critical high fidelity of the growing chain is achieved with controlled density on solid surfaces. (Maskos, U. et al., Nucleic Acids Res., 20, 1679-1684 (1992); Shchepinov, M. S. et al., Nucleic Acids Res., 25, 1155-1161 (1997); Leproust, E. et al., Nucleic Acids Res. 29, 2171-2180 (2001)). High fidelity synthesis requires high yield reactions and the subsequent capping (termination) of the reactive groups so that the reactive groups do not further react until synthesis is complete or the desired deprotection is achieved in a specific reaction step. The controlled density of the molecules synthesized on a support requires a means for placing a certain number of reactive groups on the support regardless of the number of the reactive groups on the original surface. In some cases, a capping reaction using a capping agent is necessary after the reaction to prevent the capping reagent from reacting concomitantly. This becomes more important in synthesis where a monolayer of surface molecules are made.
A common platform for micro-chemical and biological experiments is planar or microscopically planar surfaces. Among these, glass plates (e.g. microscope slides, which are borosilicate glass) or beads are readily available, easy to handle and commonly used. The solid surfaces often used are silicon oxide (Si/SiO2) based, polymeric, or nitrocellulose membrane types. These surface groups do not have ordered structures like those derivatized on Si/SiO2 crystalline silicon surfaces processed in the clean room environments of the semiconductor and micro-electronics industries. In the last few years chemical reactions on glass plate surfaces have been extensively investigated in an effort to understand and optimize synthesis and binding assays on these surfaces.
In addition to factors that affect conventional reactions, such as concentrations and stoichiometric ratios of reagents, specific concerns relate to micro-scale reactions on solid surfaces. These include, for example, the reactivity of surface functional groups, accessibility of the reactants bound to a surface, effective concentration or density of surface molecules and surface microstructures. For oligonucleotide syntheses, earlier studies addressed questions related to bulk solid support materials, such as failure sequences (n-1 sequences where n is the length of the desired sequence) on controlled porous glass (CPG). (Fearon, K. L. et al., Nucleic Acids Res., 23, 2754-2761 (1995); Temsamani, J. et al., Nucleic Acids Res. 23, 1841-1844 (1995); Iyer, R. P. et al., Nucleic Acid Res., 14, 1349-1357 (1995)). The effects of surface functional groups, pore size, chemical properties of linker molecules and linker chain length on synthesis were examined using HPLC and other conventional analytical chemical methods. (Katzhendler, J. et al., Tetrahedron 45, 2777-2792 (1989)). These studies led to the development of highly homogeneous porous glass and synthetic solid support materials containing linkers with desirable chain lengths (e.g. oligoethylene glycosyl linker) or acid/base stable chemical bonds (e.g. ether and amide linkages). In comparison to these bulk syntheses, oligonucleotide syntheses on glass plate surfaces are on the picomolar scale (0.1-1 pmol/mm2). Each spot (micro square) of a microarray of oligonucleotides contains a femtomole or less of material. These micro-quantities of material prevent reactions from being monitored using conventional methods, such as HPLC or UV. In the literature, monitoring of coupling reactions between a nucleotide phosphoramidite (monomer) and the terminal OH group of the immobilized linkers or oligonucleotides were accomplished using fluorescence (FR) measurements. (Leproust, E. et al., Nucleic Acids Res., 29, 2171-2180 (2001); LeProust, E. et al., J. Comb. Chem. 2, 349-354 (2000)). Usually fluorescein phosphoramidites are reacted with the surface terminal OH groups to form fluorescein-terminated oligonucleotides. The intensities of fluorescence emission (FRE) measured following each coupling step are considered proportional to the yields of the coupling reactions. The step-wise yields and the purity of the oligonucleotides synthesized are calculated from these FRE measurements. Using this approach, the efficiency of parallel oligonucleotide synthesis using photolithography and photolabile protection groups is reported to be in the range 82-97%. (Pirrung, M. C. et al., J. Org. Chem., 60, 6270-6276 (1995); McGall, G. H. et al., J. Am. Chem. Soc., 119, 5081-5090 (1997); Beier, M. et al., Nucleic Acids Res., 27, 1970-1977 (1999)).
A major cause of lower fidelity synthesis on glass plates is due to the particularly inefficient reactions of the various reagents with the functional groups close to glass plate surfaces. A conventional capping reagent, such as acetic anhydride (Glen Research, Sterling, Va.), for oligonucleotide synthesis especially gives low reaction yields when the reaction sites are close to the surface. Thus, unreacted and uncapped functional groups subsequently react with the nucleophosphoramidites, and the capping and coupling reaction cycles are repeated. The capped sequences are failure sequences which are shorter than full length sequences with the missing residues being at the end closest to the surface. The uncapped and subsequently reacted sequences are also shorter than the full length sequence but they are truncated at the end attached to the surface; these sequences contain deletions of certain residues at the step where coupling and capping failed as shown in FIG. 1.
There are additional problems due the presence of reactive groups on support, such as OH, NH2, or CO2H groups. The affinity of these groups to proteins, nucleic acids, and other molecules in biological samples causes non-specific adhering and interference with measuring or detecting the specific binding of these biomolecules to their substrate molecules. Non-specific adhering in the binding assays is the origin of high background signal reading, such as fluorescence intensities. This reduces the sensitivity and dynamic range of the devices used for such analyses. It is therefore necessary to cap these reactive groups on the surface of support to reduce non-specific adhering of the various molecules.
One family of polyether molecules has been extensively studied and applied to fields such as industrial processing materials, drug delivery formulation reagents, surface materials, synthesis supports, separation supports, peptide/protein modifiers, and as gradients of the various biomaterials. (Poly(Ethylene Glycol): Chemistry and Biological Applications (Acs Symposium Series, No 680) by J. Milton Harris (Editor), Samuel Zalipsky (Editor), American Chemical Society Division of Polymer Chemistry, Calif.) American Chemical Society Meeting 1997 San Francisco, Zalipsky Harris). Typical compounds of the ether family of polymers include oligoethylene glycol (OEG) or oligoethylene oxide, polyethylene glycol (PEG) or polyethylene oxide, oligopropylene oxide (OPO), and polypropylene oxide (PPO). Polymers of ethylene glycol (EG) comprise polyether linkages and the repeating unit is —(OCH2CH2)—. Polymers of propylene oxide (PO) comprise polyether linkages and the repeating unit is —(OCH2CH2CH2)—.
PEG molecules (44 Da per monomer unit and a length of ˜3.9 Å per repeating unit in an extended conformation) are amphiphilic in nature, i.e., they possess hydrophilic and hydrophobic properties that allow their solubility in aqueous or organic solvents. PEG dissolves in water to form a biphasic solution with PEG on the top layer and structured water molecules surrounding the PEG chain. Historically, PEG, and especially higher molecular weight PET, is known to be a salt-out reagent that causes protein precipitation. (Arakawa T. et al., Biochemistry, 24, 6756-6762 (1985)). This property can be favorably used to prepare a protein-repellant surface. Presently, there is a need for non-adhesive surfaces for protein assays. In light of this need, various PEG surfaces, such as PEG grafted silicon surfaces, have been prepared. (Zhu, X.-Y. et al., Langmuir, 17, 7798-7803 (2001)).
Shorter PEG molecules or OEG have been used as spacer or tethers in biopolymer conjugates, such as those used in preparation of oligonucleotide-PEG-oligonucleotide conjugates. (Knoll, E. et al., Anal. Chem. 76, 1156-1164 (2004)). In these applications, the OEG used has the general structure of X—(OCH2CH2)n—Y, where X and Y are reactive groups that can be attached to the molecules to form a conjugate compound and n is the number of repeating units. As an example, in an OEG used as spacer (Glen Research, Sterling, Va.), X is a phosphoramidite, Y is ODMT (DMT is 4,4′-dimethoxytrityl), and n is six. The phosphoramidite reacts with an OH group, such as the 5′-OH of an oligonucleotide, to form an internucleotide phosphate linkage after the oxidation reaction. DMT can then be easily removed to give an OH group which can couple with a nucleophosphoramidite to form internucleotide phosphate linkage after the oxidation reaction. The final product of these reactions has the structure 5′-oligonucleotide-(OCH2CH2)6—O-oligonucleotide-3′ which is referred to as conjugated oligonucleotides or tethered oligonucleotides. The compounds on either side of the spacer do not have to be identical or even of the same type of molecule. For instance, a peptide can be tethered with an oligonucleotide to give a peptide-oligonucleotide conjugate; or, an oligonucleotide can be tethered to a surface reactive group to be immobilized on surface.